The present invention is related to results obtained from research on deoxyribonuclease (DNase), a phosphodiesterase that is capable of hydrolyzing polydeoxyribonucleic acid. It relates generally to the separation of several forms of said DNase, to these forms per se, to pharmaceutical compositions by which their utility can be exploited clinically, and to methods of using these DNases and compositions thereof.
DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. DNase has been purified from various species to various degrees. The complete amino acid sequence for a mammalian DNase was first made available in 1973. See e.g., Liao, et al., J. Biol. Chem. 248:1489 (1973).
DNase has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions in such diseases as pneumonia and cystic fibrosis, thereby aiding in the clearing of respiratory airways. See e.g., Lourenco, et al., Arch. Intern. Med. 142:2299 (1982); Shak, et al., Proc. Nat. Acad. Sci. 87:9188 (1990); Hubbard, et al., New Engl. J. Med. 326:812 (1992).
DNA encoding human DNase I has been isolated and sequenced and that DNA has been expressed in recombinant host cells, thereby enabling the production of human DNase in commercially useful quantities. See e.g., Shak, et al., Proc. Nat. Acad. Sci. 87:9188-9192 (1990). Recombinant human DNase (rhDNase) has been found to be useful clinically, especially in purified form such that the DNase is free from proteases and other proteins with which it is ordinarily associated in nature. See e.g., Hubbard, et al., New Engl. J. Med. 326:812 (1992).
The means and methods by which human DNase can be obtained in pharmaceutically effective form is described in the patent applications cited above. Various specific methods for the purification of DNase are known in the art. See e.g., Khouw, et al., U.S. Pat. No. 4,065,355 (issued Dec. 27, 1977); Markey, FEBS Letters 167:155 (1984); Nefsky, et al., Eur. J. Biochem. 179:215 (1989).
Although it was not appreciated at the time the above-referenced patent applications were filed, the DNase product obtained from cultures of recombinant host cells typically comprises a mixture of deamidated and non-deamidated forms of DNase. The existence of deamidated forms of DNase remained unappreciated notwithstanding that the phenomenon of deamidation of asparagine and glutamine residues in some proteins is known. See e.g., Eipper et al., Ann. Rev. Physiol. 50:333 (1988); Kossiakoff, Science 240:191 (1988); Bradbury et al., Trends in Biochem. Sci. 16:112 (1991); and Wright, Protein Engineering 4:283 (1991);
The present invention is predicated upon the previously unappreciated fact that recombinant human DNase may exist as a mixture of deamidated and non-deamidated forms. Using the methods of the present invention, it has been found that deamidated human DNase is less active enzymatically than non-deamidated human DNase. Thus, the presence of the deamidated DNase and non-deamidated DNase together in a mixture, and the potential for further deamidation occurring, such as has been found to occur upon in vitro storage of preparations of human DNase, may complicate efforts to provide consistent uniformity in a DNase product being administered clinically. Therefore, as the existence and characteristics of deamidated DNase were not known prior to the present invention, the methods for identifying deamidated DNase and separating it from preparations of DNase in which it may be found were unobvious at the time this invention was made.
The present invention is directed to processes for separating the deamidated and non-deamidated human DNase forms from a mixture thereof. This process in preferred embodiments comprises subjecting the mixture to chromatography using a resin, or other support medium, having bound thereto a cationic polymer such as heparin or a non-hydrolyzable deoxyribonucleic acid (DNA) analog, or chromatography using a so-called tentacle cation exchange resin. The present invention also is directed to the use of those chromatographic methods with non-human DNases, such as bovine DNase.
The present invention also is directed to deamidated human DNase as a purified product, substantially free of non-deamidated human DNase.
The present invention also is directed to non-deamidated human DNase as a purified product, substantially free of deamidated human DNase. It has been found herein that purified non-deamidated human DNase is fully enzymatically active as compared with deamidated human DNase.
The present invention also is directed to pharmaceutical compositions consisting of either purified deamidated human DNase or purified non-deamidated human DNase as the active principle, optionally together with a pharmaceutically acceptable excipient.
The present invention also is directed to a method comprising administering a therapeutically effective amount of purified deamidated human DNase or purified non-deamidated human DNase for the treatment of a patient, for example those having an accumulation of viscous, DNA-containing material. The administration of such purified DNases preferably is effected by direct inhalation into the lungs.
The present invention is particularly directed to a method of treating a patient having a pulmonary disease such as chronic bronchitis, cystic fibrosis, or emphysema, that comprises administering a therapeutically effective amount of purified non-deamidated human DNase, preferably directly into the airway passages.
The present invention also is directed to pharmaceutical compositions comprising non-deamidated human DNase that are disposed within a plastic vial, optionally in the presence of a pharmaceutically acceptable excipient.